Uplansapo 60x 1.35 oil

U2OS cells stably expressing RFP-53BP1 in 35 mm glass-bottom dishes (WillCo-dish) were transfected with 1 μg of expression plasmids coding for GFP-UCHL3 (wild-type) or (C95A mutant) with Mirus TransIT-LT1 and further cultured for 48 hours in the presence of 10 μM 5-Bromo-2′-deoxyuridine (BrdU). On the day of analysis, the media was replaced with phenol red-free DMEM (Sigma-Aldrich) supplemented with 10% FBS, penicillin, streptomycin and 25 mM HEPES buffer (pH 7.0–7.6, Sigma-Aldrich). DNA damage was induced by irradiating cells through a UPlanSApo 60x/1.35 oil objective lens with UV-A laser (405 nm) using a IX81 confocal microscope (Olympus) equipped with a 37 °C heating stage (Ibidi). The laser output was set at 400 mW with 50 scans of 10 msec/pixel. Prior to DNA damage induction and 15 min after damage induction, images were taken and analyzed by using FluoView 1000 software (Olympus). The signal intensity was quantified by using ImageJ.

Cleaned or PEI-coated cover glasses were placed in a sealed PTFE-ring and each surface was covered with 1 ml of a 10% ethanol (AppliChem, Germany) HEPES-buffer mixture pH = 7.0. 100 μl of a suspension containing COOH-functionalized particles (section 2.2) was added dropwise afterwards. After a period of 15 min, sedimentation of the particles was completed and the probes and their corresponding radial profiles were imaged using an inverted microscope (Olympus IX73, Germany) with an integrated halogen lamp for bright-field microscopy. To obtain the respective interference reflection patterns, samples were illuminated by a monochromatic 530 nm collimated LED (M530L2-C1, Thorlabs, Germany). An Olympus 60x, NA (numerical aperture) 1.35 oil-immersion objective (UPlanSApo 60x 1.35 oil, Olympus, Germany) was used in concert with a quarter waveplate (WPMQ05M-532, Thorlabs GmbH, Germany), placed on the microscope’s breadboard between objective and sample, as well as additional polarizers to avoid internal reflections [19 (link)]. Images were captured with a monochrome CCD camera (DMK 23U274, ImagingSource, Germany) using μManager microscopy software. All datasets were recorded applying an exposure time of 50 ms and stored in tagged image file format (tiff). Besides the methods explained in the following sections, no image processing was performed.